Methods for treating glucose metabolic disorders

ABSTRACT

In one aspect, the invention relates to methods for treating a disease or condition associated with abnormal glucose metabolism. In another aspect, the invention relates to a method for reducing incidence or progression of insulin-dependent diabetes mellitus. The methods include orally administering to a mammal in need thereof an effective amount of a composition, which includes a naturally occurring material derived from eggshell, eggshell membrane, or a combination thereof.

CROSS REFERENCE RELATED APPLICATION

The present invention asserts priority to U.S. Provisional ApplicationSer. No. 61/323,303, filed on Mar. 12, 2010, the contents of which areincorporated herein by reference.

INCORPORATION BY REFERENCE

The present invention incorporates by reference the disclosure of U.S.patent application Ser. No. 11/943,169, filed on Nov. 20, 2007, in itsentirety.

BACKGROUND

Diabetes is one of the most prevalent chronic diseases in the UnitedStates, and it is a leading cause of death. Persons with diabetes are atrisk for major complications, including diabetic ketoacidosis, end-stagerenal disease, diabetic retinopathy and amputation. There are also ahost of less directly related conditions, such as hypertension, heartdisease, peripheral vascular disease and infections, for which personswith diabetes are at substantially increased risk. Accordingly, newmethods of treating diabetes and diseases or conditions associated withabnormal glucose metabolism are needed.

SUMMARY OF THE INVENTION

These objectives, among others, are addressed by the present invention.

In one aspect, the invention relates to a method for treating a diseaseor condition associated with abnormal glucose metabolism in a mammal inneed thereof. The method includes orally administering to the mammal aneffective amount of a composition, which comprises a naturally occurringmaterial derived from eggshell membrane.

In one embodiment, the disease or condition is insulin resistance.

In one embodiment, the disease or condition is glucose intolerance.

In one embodiment, the disease or condition is glucosenon-responsiveness.

In one embodiment, the disease or condition is diabetes mellitus.

In one embodiment, the effective amount is effective to achieve at leasta 0.2 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 1 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 2 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 3 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 5 mg/dL.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 10 mg/dL.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 20 mg/dL.

In one embodiment, the naturally occurring material is selected from thegroup consisting of a hexosamine, glycosaminoglycan, hyaluronic acid,sialic acid, collagen, elastin, other egg proteins or glycoproteins,lysozyme, ovotransferrin, lysyl oxidase, and/or a combination thereof.

In one embodiment, the hexosamine is glucosamine and/orN-acetylglucosamine.

In one embodiment, the glycosaminoglycan is chondroitin sulfate,dermatan sulfate, keratin sulfate, heparin sulfate, and/or a combinationthereof.

In one embodiment, the naturally occurring material is water soluble.

In one embodiment, the naturally occurring material is water insoluble.

In one embodiment, the naturally occurring material comprises thefollowing in weight percent:

-   a. about 0.25 to about 5 wt % hexosamine;-   b. about 0.3 to about 3 wt % glycosaminoglycan;-   c. about 0.5 to about 5 wt % hyaluronic acid;-   d. about 0.01 to about 2 wt % sialic acid; and/or-   e. about 5 to about 20 wt % collagen.

In one embodiment, the naturally occurring material is an eggshellmembrane powder.

In one embodiment, the naturally occurring material is an eggshellmembrane extract.

In one embodiment, the naturally occurring material is an eggshellmembrane hydrosylate.

In one embodiment, the naturally occurring material is an eggshellmembrane isolate.

In one embodiment, the mammal is insulin-dependent.

In one embodiment, the mammal has a fasting blood glucose concentrationat least 100 mg/dL and at most 125 mg/dL.

In one embodiment, the mammal is diabetic.

In another aspect, the invention relates to a method for reducingincidence or progression of insulin-dependent diabetes mellitus in amammal in need thereof. The method includes orally administering to themammal an effective amount of a composition, which comprises a naturallyoccurring material derived from eggshell membrane.

In one embodiment, the disease or condition is insulin resistance.

In one embodiment, the disease or condition is glucose intolerance

In one embodiment, the disease or condition is glucosenon-responsiveness.

In one embodiment, the disease or condition is diabetes mellitus.

In one embodiment, the effective amount is effective to achieve at leasta 0.5 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 1 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 2 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 3 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 5 mg/dL.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 10 mg/dL.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 20 mg/dL.

In one embodiment, the naturally occurring material is a therapeuticallyactive material selected from the group consisting of eggshell membranepowder, an eggshell membrane hydrolysate, an eggshell membrane isolate,and combinations thereof.

In one embodiment, the naturally occurring material is selected from thegroup consisting of a hexosamine, glycosaminoglycan, hyaluronic acid,sialic acid, collagen, elastin, other egg proteins or glycoproteins,lysozyme, ovotransferrin, lysyl oxidase, and/or a combination thereof.

In one embodiment, the hexosamine is glucosamine and/orN-acetylglucosamine.

In one embodiment, the glycosaminoglycan is chondroitin sulfate,dermatan sulfate, keratin sulfate, heparin sulfate, and/or a combinationthereof.

In one embodiment, the naturally occurring material is water soluble.

In one embodiment, the naturally occurring material is water insoluble.

In one embodiment, the naturally occurring material is eggshell membranepowder.

In one embodiment, the naturally occurring material is an eggshellmembrane extract.

In one embodiment, the naturally occurring material is an eggshellmembrane hydrolysate.

In one embodiment, the naturally occurring material is an eggshellmembrane isolate.

In one embodiment, the mammal is insulin-dependent.

In one embodiment, the mammal has a fasting blood glucose concentrationat least 100 mg/dL and at most 125 mg/dL.

In one embodiment, the mammal is diabetic.

In one embodiment, the naturally occurring material comprises thefollowing in weight percent:

-   a. about 0.25 to about 5 wt % hexosamine;-   b. about 0.3 to about 3 wt % glycosaminoglycan;-   c. about 0.5 to about 5 wt % hyaluronic acid;-   d. about 0.01 to about 2 wt % sialic acid; and/or-   e. about 5 to about 20 wt % collagen.

In a further aspect, the invention relates to a method for treating adisease or condition associated with abnormal glucose metabolism in amammal in need thereof. The method includes orally administering to themammal an effective amount of a composition, which comprises a naturallyoccurring material derived from fowl eggshells.

In one embodiment, the disease or condition is insulin resistance.

In one embodiment, the disease or condition is glucose intolerance

In one embodiment, the disease or condition is glucosenon-responsiveness.

In one embodiment, the disease or condition is diabetes mellitus.

In one embodiment, the effective amount is effective to achieve at leasta 0.2 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 1 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 2 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to achieve at leasta 3 percentage point decrease in hemoglobin A1c.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 5 mg/dL.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 10 mg/dL.

In one embodiment, the effective amount is effective to decrease afasting blood glucose concentration in said mammal by at least 20 mg/dL.

In one embodiment, the naturally occurring material is selected from thegroup consisting of a hexosamine, glycosaminoglycan, hyaluronic acid,sialic acid, collagen, elastin, other egg proteins or glycoproteins,lysozyme, ovotransferrin, lysyl oxidase, and/or a combination thereof.

In one embodiment, the hexosamine is glucosamine and/orN-acetylglucosamine.

In one embodiment, the glycosaminoglycan is chondroitin sulfate,dermatan sulfate, keratin sulfate, heparin sulfate, and/or a combinationthereof.

In one embodiment, the naturally occurring material is water soluble.

In one embodiment, the naturally occurring material is water insoluble.

In one embodiment, the naturally occurring material comprises thefollowing in weight percent:

-   a. about 0.25 to about 5 wt % hexosamine;-   b. about 0.3 to about 3 wt % glycosaminoglycan;-   c. about 0.5 to about 5 wt % hyaluronic acid;-   d. about 0.01 to about 2 wt % sialic acid; and/or-   e. about 5 to about 20 wt % collagen.

In one embodiment, the naturally occurring material is an extract.

In one embodiment, the naturally occurring material is a hydrolyzate.

In one embodiment, the naturally occurring material is an isolate.

In one embodiment, the mammal is insulin-dependent.

In one embodiment, the mammal has a fasting blood glucose concentrationat least 100 mg/dL and at most 125 mg/dL.

In one embodiment, the mammal is diabetic.

DETAILED DESCRIPTION Method for Treating a Disease or ConditionAssociated with Abnormal Glucose Methabolism

In one aspect, the invention relates to a method for treating a diseaseor condition associated with abnormal glucose metabolism in a mammal inneed thereof. The method includes orally administering to the mammal aneffective amount of a composition. The composition includes a naturallyoccurring material derived from eggshell membrane or from fowleggshells.

A disease or condition associated with abnormal glucose metabolismrefers to a disease or condition wherein the mammal has abnormal glucosemetabolism, as compared to a respective normal mammal having the samegender, weight, height, and/or age as the treated mammal. Typically,abnormal glucose metabolism is characterized by one or a combination ofthe following factors: abdominal obesity, insulin resistance, glucoseintolerance, hypertension, above-normal blood sugar (serum glucose)concentration, and above-normal hemoglobin A1c percentage point.

Examples of a disease or condition associated with abnormal glucosemetabolism include insulin resistance, glucose intolerance, glucosenon-responsiveness, diabetes mellitus, and/or a combination thereof.

Insulin resistance refers to a failure of the body to respond normallyto insulin. Normal fasting insulin levels are considered to be <10μIU/mL, although there is considerable variability within thepopulation. In subjects suffering from insulin resistance, high bloodsugar levels stimulate production of insulin. When a subject is unableto normally process excess insulin, insulin levels rise. Eventually, thesubject has high blood sugar levels (hyperglycemia) and high insulinlevels (hyperinsulemia). Under these conditions, insulin loses itsability to control fat metabolism, and excessive fats enter thebloodstream (hyperlipidemia). Hyperlipidemia contributes to high bloodpressure, heart disease and stroke. Other disorders of insulinresistance, include, but are not limited to, dyslipidemia, (includingdiabetic dyslipidemia) and full-blown Type 2 diabetes, juvenile diabetesand gestational diabetes.

Common forms of insulin resistance include, for example, skeletal muscleinsulin resistance, hepatic insulin resistance and adipose tissueinsulin resistance. Metabolic syndrome is a clinical diagnosisconsidered to be a form of insulin resistance and is generally definedas having any three or more of the following conditions: 1) waistmeasurement of 40 inches or more for men and 35 inches or more forwomen, 2) triglyceride levels of 150 milligrams per deciliter (mg/dL) orabove, or taking medication for elevated triglyceride levels, 3) HDLcholesterol level below 40 mg/dL for men and below 50 mg/dL for women,or taking medication for low HDL levels, 4) blood pressure levels of130/85 or above, or taking medication for elevated blood pressurelevels, or 5) fasting blood glucose levels of 100 mg/dL or above, ortaking medication for elevated blood glucose levels.

The degree of insulin resistance may vary amongst subjects. Insulinresistance typically precedes the development of adult onset diabetes.Insulin resistance is often a precursor to Type 2 diabetes. However,mild or even severe insulin resistance may be found in individuals whowill never develop diabetes. Genetic factors contribute to this normalvariation in insulin resistance.

Glucose intolerance refers to insufficiency of an insulin secretionresponse in a mammal due to glucose load and/or reduction of insulinaction in skeletal muscles or adipose tissues. Accordingly, the mammalis unable to utilize glucose in blood circulation. In some cases,glucose intolerance is caused by insulin resistance. A glucoseintolerant subject may have a serum glucose concentration greater thanabout 100 mg/dL, greater than about 110 mg/dL, or greater than about 120mg/dL, as compared to a control serum glucose concentration.

Glucose intolerance is a condition that may precede the onset ofdiabetes and it may be associated with various metabolic diseases orconditions, such as obesity, hypertension, hypertriglyceridemia, etc.Continuous glucose intolerant conditions may induce onset of diabetesand may also enhance the progress of diabetes. Therefore, treatment ofglucose intolerance is considered effective in reducing an incidence orprogression of diabetes.

Glucose non-responsiveness refers to the complete inability of cells,islets or mammals to respond to treatment with or administration ofglucose, as well as decreased responsiveness to glucose (e.g., by cellsthat do not produce sufficient levels of insulin in response to glucoseor that require significantly higher levels of glucose to respond atnormal levels).

Diabetes or diabetes mellitus is a disease that occurs when the bodycannot make use of the glucose in the blood for energy because eitherthe pancreas is not able to make enough insulin or the insulin that isavailable is not effective. There are two main types of diabetesmellitus: insulin-dependent (type 1) and noninsulin-dependent (type 2 oradult onset diabetes). A third type of diabetes is gestational diabetesthat develops only in pregnant women.

Method for Reducing Incidence or Progression of Insulin-DependentDiabetes Mellitus

In another aspect, the invention relates to a method for reducingincidence or progression of insulin-dependent diabetes mellitus in amammal in need thereof. The method includes orally administering to themammal an effective amount of a composition. The composition includes anaturally occurring material derived from eggshell membrane or from fowleggshells.

Reducing incidence or progression of the disease or condition refers toalleviation of symptoms, diminishment of extent of disease or condition,stabilized (i.e., not worsening) state of disease or condition, delay orslowing of disease progression, amelioration or palliation of thedisease state and remission (whether partial or total), whetherdetectable or undetectable. For example, mammals with abnormal glucosemetabolism typically have a significant risk of developing diabetes. Thepresent invention reduces incidence or progression of the disease orcondition, for example, by preventing a transition from abnormal glucosemetabolism to insulin-dependent diabetes mellitus in a mammal.

In insulin-dependent diabetes (IDDM; type I diabetes), the pancreasmakes little or no insulin because the insulin-producing beta cells havebeen destroyed. IDDM may occur at any age. Current treatment typicallyincludes daily insulin injections or use of an insulin pump, a planneddiet and regular exercise, and daily self-monitoring of blood glucose.If the level of insulin is too low for a long period of time, the bodybegins to break down its stores of fat for energy. The break down of fatstores causes the body to release fatty acids, which are then convertedinto ketone bodies or ketoacids that are toxic at high levels. Theresult is called ketoacidosis, a severe condition that may put a personinto a coma if not treated promptly.

Mammal

The present invention is suitable for both medical and veterinary uses.Suitable subjects include, but are not limited to, mammalian and aviansubjects; preferably, mammalian subjects; more preferably humans,monkeys, pigs, cattle, dogs, horses, cats, sheep, mice, rats, guineapigs, rabbits, and goats, and are most preferably human subjects. Themammal may be male or female, of any age.

A mammal in need of the methods of the invention includes those alreadysuffering from the disease or condition, as well as those prone todevelop the disease or condition, and those in whom the condition ordisorder is to be prevented.

In one embodiment, the mammal is insulin-dependent. An insulin-dependentmammal is one that has been prescribed insulin injections or use of aninsulin pump, and/or equivalent treatments.

In another embodiment, the mammal is pre-diabetic. A mammal that ispre-diabetic has a glucose metabolic state that is intermediate betweennormal glucose homeostasis and metabolism and a metabolic state observedin a respective mammal having diabetes. For example, a mammal that ispre-diabetic typically has a fasting blood glucose concentration atleast 100 mg/dL and at most 125 mg/dL typically indicates pre-diabetes.As another example, a mammal that is pre-diabetic typically has ahemoglobin A1c level that is about 6.5% to about 7.0% of totalhemoglobin.

In a further embodiment, the mammal is diabetic, suffering from diabetesas described above. A mammal that is diabetic typically has a glucosemetabolic state that is abnormal. For example, a mammal that is diabetictypically has a fasting blood glucose concentration at least about 125mg/dL or greater. As another example, a mammal that is diabetictypically has a hemoglobin A1c level that is at least about 7.0% oftotal hemoglobin or greater.

Administration

The methods of the invention include orally administering to a mammal inneed thereof an effective amount of the composition. For oraladministration, the composition may be in the form of any unit dosagevehicle, such as a capsule or tablet, or in the form of powder, liquid,or contained in any convenient food item. The composition may alsoinclude pharmaceutically or nutraceutically active ingredients, fillers,binders, lubricants, flow agents, colorants, or other processing agents.

In one embodiment, the effective amount of the composition administeredin accordance with the method of the invention is any amount effectivefor treating a disease or condition associated with abnormal glucosemetabolism. In another embodiment, the effective amount of thecomposition administered in accordance with the method of the inventionis any amount effective for reducing incidence or progression ofinsulin-dependent diabetes mellitus.

The administered amount of the composition will vary according tonumerous factors that are well known in the art, such as the disease orcondition to be treated or reduced, the route of administration, theparticular mammal to be treated, the status of the disease or conditionin the subject, etc. The appropriate amount of the composition canreadily be determined by those skilled in the art.

For example, the effective amount of the composition may be any amountthat is sufficient to achieve a decrease in a percentage point ofhemoglobin A1c. In one embodiment, the effective amount is effective toachieve at least a 0.2 percentage point, a 1 percentage point, a 2percentage point, or a 3 percentage point decrease of hemoglobin A1c inthe subject. Methods of determining percentage point decrease ofhemoglobin A1c in a mammal are known in the art.

In healthy, non-diabetic mammals, the hemoglobin A1c level is typicallyless than about 6.5%, preferably less than about 6.0%, and morepreferably less than about 5.5% of total hemoglobin. Complications ofdiseases or conditions associated with abnormal glucose metabolism canbe reduced, delayed, or treated if the percentage of total hemoglobinA1c is maintained below 6.5%. Accordingly, in one embodiment of theinvention, the effective amount is effective to achieve a totalhemoglobin A1c level less than about 6.5%, preferably less than about6.0%, and more preferably less than about 5.5% of total hemoglobin.Consequently, in another embodiment, the effective amount is effectivefor achieving insulin-independence in an insulin-dependent mammal. See,for example, Example 2.

A mammal in need of a method of the invention may have a hemoglobin A1clevel that is greater than about 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%,9.5% of total hemoglobin or greater. A hemoglobin A1c level that isabout 6.5% to about 7.0% of total hemoglobin typically indicatespre-diabetes. A hemoglobin A1c level that is greater than about 7.0%typically indicates diabetes. Accordingly, compositions of the presentinvention may be administered to a mammal with a hemoglobin A1c levelequal or greater than about 6.5% of total hemoglobin.

As another example, the effective amount of the composition may be anyamount that is sufficient to achieve a decrease in fasting blood glucoseconcentration in the mammal. A mammal's blood glucose may be measuredafter fasting for 8 to 12 hours. In one embodiment, the effective amountis effective to decrease a fasting blood glucose concentration in themammal by at least 5 mg/dL, at least 10 mg/dL, at least 20 mg/dL.Methods of determining a decrease of fasting blood glucose concentrationin a mammal are known in the art.

In one embodiment of the invention, the effective amount is effective toachieve a normal fasting blood glucose concentration in the mammal. Amammal with normal fasting blood glucose typically has a fasting bloodglucose concentration that is at least 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, or 110 mg/dL. A mammal with normal fastingblood glucose typically has a fasting blood glucose concentration thatis less than 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110mg/dL. Any minimum value may be combined with a maximum value in orderto describe a range of normal fasting blood glucose concentration for amammal.

As is known in the art, a mammal having normal fasting blood glucoseconcentration is typically insulin-independent. Accordingly, in anotherembodiment, the effective amount is effective for achievinginsulin-independence in an insulin-dependent mammal. See, for example,Example 2.

A mammal in need of a method of the invention may have a fasting bloodglucose concentration that is greater than about 100, 105, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125mg/dL. A fasting blood glucose concentration at least 100 mg/dL and atmost 125 mg/dL typically indicates pre-diabetes. A fasting blood glucoseconcentration greater than 125 mg/dL indicates diabetes. Accordingly,compositions of the present invention may be administered to a mammalwith fasting blood glucose concentrations between 110 mg/dL and 125mg/dL. For example, the mammal in need thereof may have a fasting bloodglucose concentration of 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125 mg/dL, or greater.

In one embodiment, administration of a composition of the inventiondecreases fasting blood glucose concentrations to less than 125, 124,123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, andpreferably below 110 mg/dL.

The methods of the present invention may be combined with any knowntreatment or prevention regimen for diseases or conditions associatedwith abnormal glucose metabolism and diabetes mellitus. The methods andcompositions of the invention may be simultaneously, separately orsequentially administered with any known treatment regimens.

In a preferred embodiment, a composition of the present invention may beadministered long-term for the methods of the invention. In this regard,the composition may be administered for a period of at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or more days. The compositions may alsobe administered for an administration period of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 or more months. The compositions may also beadministered for an administration period of at least 1, 2, 3, 4, 5, 6,7 8, 9, 10 or more years. During the administration period, thecomposition may be administered once daily, twice daily, thrice daily,or periodically, such as every other day, etc.

The composition of the present invention may also be administeredintermittently for the methods of the invention. For example, thecomposition may be administered for an administration period of 1, 2, 3,4, 5, or more weeks, followed by a period of discontinuance, followed byan administration period of 1, 2, 3, 4, 5 or more weeks.

Composition

The composition used in the methods of the invention includes anaturally occurring material. The terms, “natural material,” “naturallyoccurring material” or “naturally occurring active material” derivedfrom eggshell, eggshell membrane, or a combination thereof refer tomaterial that contains a significant amount of at least one ingredientor component of the eggshell, eggshell membrane, or a combinationthereof that is substantially unaltered from an untreated or unprocessedeggshell, eggshell membrane, or a combination thereof, respectively.“Substantially unaltered” refers to a characteristic of a selected ordesired ingredient(s) or component(s), having substantially retainedits/their physical and/or chemical characteristics and is/are notsignificantly decomposed, digested or cleaved. However, other componentsor ingredients may be altered in certain isolates or hydrolysates. Forexample, hydrolysates prepared by enzyme treatment may result innaturally occurring proteins being at least partially digested.

Preferably, the majority of the naturally occurring ingredients found inthe eggshell, eggshell membrane, or a combination thereof aresubstantially unaltered and, more preferably, substantially all of thenaturally occurring ingredients are substantially unaltered. Althoughthe physical characteristics of individual components of the eggshell,eggshell membrane, or a combination thereof remain substantiallyunaltered, the overall composition or amounts of different componentscan be altered depending on the desired composition for a particularisolate, extract or hydrolysate.

In one embodiment, the composition may contain a material derived fromeggshell, eggshell membrane, or a combination thereof that is processedto be rich in water-soluble fractions of the respective eggshell,eggshell membrane, or a combination thereof that is rich in hyaluronicacid.

In another embodiment, the composition may contain a material derivedfrom eggshell, eggshell membrane, or a combination thereof that is richin water-insoluble fractions and/or is rich in certain collagens.

The material may also contain a specific component selected from thegroup consisting of a hexosamine, chondroitin sulfate, hyaluronic acid,collagen, other proteins, and combinations thereof. In one embodiment,the material includes at least about 10 wt % and at most about 80 wt %of other egg proteins or glycoproteins derived from eggshell, eggshellmembrane, or a combination thereof, based on total weight of thematerial. It is also contemplated that the material derived from theeggshell, eggshell membrane, or a combination thereof can be processedto be rich in specific type(s) of collagen and/or proteins, dependingupon the intended use.

In an embodiment of the invention, the material contains at least about0.1 wt %, 0.25 wt %, 0.5 wt %, 1 wt %, 2 wt %, 3 wt %, 4 wt %, or about4.5 wt % hexosamine, and the material contains at most about 10 wt %, 9wt %, 8 wt %, 7 wt %, 6 wt %, or about 5 wt % hexosamine, based on thetotal weight of the material. Any minimum value may be combined with amaximum value in order to describe a range. Preferably, the materialincludes at least about 0.25 wt % and at most about 5 wt % hexosamine.

Examples of hexosamine include glucosamine, N-acetyl-D-glucosamine,glucosamine hydrochloride, glucosamine sulfate, and combinationsthereof. Preferably, the hexosamine is glucosamine orN-acetyl-D-glucosamine.

In another embodiment, the material contains at least about 0.1 wt %,0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 0.6 wt %, 0.7 wt %, 0.8 wt %,0.9 wt %, or about 1 wt % glycosaminoglycan, and the material containsat most about 5 wt %, 4 wt %, 3 wt %, 2.5 wt %, 2.0 wt %, or about 1.5wt % glycosaminoglycan, based on the total weight of the material. Anyminimum value may be combined with a maximum value in order to describea range. Preferably, the material includes at least about 0.3 wt % andat most about 3% glycosaminoglycan.

Examples of a glycosaminoglycan include chondroitin sulfate, dermatansulfate, keratan sulfate, heparan sulfate, and combinations thereof.

In yet another embodiment, the material contains at least about 0.1 wt%, 0.2 wt %, 0.3 wt %, 0.4 wt %, 0.5 wt %, 1 wt %, 1.5 wt %, 2.0 wt %,2.5 wt %, 3.0 wt %, 3.5 wt %, 4 wt % or about 4.5 wt % hyaluronic acid,and the material contains at most about 10 wt %, 9 wt %, 8 wt %, 7 wt %,6 wt %, or about 5 wt % hyaluronic acid, based on the total weight ofthe material. Any minimum value may be combined with a maximum value inorder to describe a range. Preferably, the material includes at leastabout 0.5 wt % and at most about 5% hyaluronic acid. In a preferredembodiment, the ratio of hyaluronic acid to the other naturallyoccurring materials in the composition is in the range of about 10:0.1to about 0.1:10.

In a further embodiment, the material contains at least about 0.01 wt %,0.02 wt %, 0.03 wt %, 0.04 wt %, 0.05 wt %, 0.06 wt %, 0.07 wt %, 0.08wt %, 0.09 wt %, or about 0.1 wt % sialic acid, and the materialcontains at most about 3 wt %, 2.5 wt %, 2.0 wt %, 1.5 wt %, 1.0 wt % orabout 0.5 wt % sialic acid, based on the total weight of the material.Any minimum value may be combined with a maximum value in order todescribe a range. Preferably, the material includes at least about 0.01wt % and at most about 2 wt % sialic acid.

In yet a further embodiment, the material contains at least about 1 wt%, 2 wt %, 3 wt %, 4 wt %, or 5 wt % collagen, and the material containsat most about 40 wt %, 35 wt %, 30 wt %, 25 wt %, 20 wt %, 15 wt %, orabout 10 wt % collagen, based on the total weight of the material. Anyminimum value may be combined with a maximum value in order to describea range. Preferably, the material includes at least about 5 wt % and atmost about 20 wt % collagen. The collagen can be a type selected fromthe group consisting of Type I collagen, Type V collagen, Type Xcollagen, and combinations thereof. In a preferred embodiment, the ratioof collagen to the other naturally occurring materials is in the rangeof about 10:0.1 to about 0.1:10.

The naturally occurring material may be derived from eggshell, eggshellmembrane, or a combination thereof.

Eggshell

In one embodiment of the invention, the method includes orallyadministering to a mammal in need thereof an effective amount of acomposition, which includes a naturally occurring material derived fromeggshells, preferably fowl eggshells. Eggshell refers to the calcareousportion of eggs from which the eggshell membrane is removed. Suitableeggshells may be obtained from chickens or other birds and fowl, suchas, for example, ducks, emu, ostrich, dove, quail, grouse, goose,turkey, ostrich, game hen, squab, pheasant, etc.

In one embodiment, the naturally occurring material derived fromeggshell is an eggshell powder. For example, the eggshell material maybe pulverized to produce an eggshell powder having a particle sizebetween 100-500 microns. Powdering or pulverizing methods are known inthe art.

In another embodiment, the naturally occurring material derived fromeggshell is an eggshell extract. In yet another embodiment, thenaturally occurring material derived from eggshell is an eggshellhydrolysate. In a further embodiment, the naturally occurring materialderived from eggshell is an eggshell isolate.

Eggshell, processed eggshell, and eggshell hydrolysates or isolatescontain naturally occurring materials derived from eggshell. Thesematerials can be selected from the group consisting of a hyaluronicacid, hexosamine, chondroitin sulfate, collagen and combinationsthereof.

The eggshell, processed eggshell, and eggshell hydrolysates and isolatesare preferably free of any animal body components or traces thereof,e.g., animal tissue, blood or body fluid components, which aredetrimental or undesirable for the contemplated use of the naturallyoccurring material or composition.

Eggshell Membrane

In one embodiment of the invention, the method includes orallyadministering to a mammal in need thereof an effective amount of acomposition, which includes a naturally occurring material derived fromeggshell membrane. Eggshell membrane is composed of two individualmembranes between egg albumin and eggshell. The membranes are primarilycomprised of protein fibers. The fibers appear to be a network orscaffold predominantly containing Type I collagen fibers that areencapsulated in a continuous mantle of proteoglycans and othermacromolecules. The thickness of the two membranes ranges from 73-114 μmin eggs from White Leghorn and New England pullets. The outer membranehas a thickness ranging from 53.2 μm to 65.5 μm in White Leghorn eggswhile the inner membrane ranges form 19.5 μm to 24.3 μm.

The proteins of shell membranes typically exhibit a high content ofarginine, glutamic acid, methionine, histidine, cystine, and proline.Eggshell membranes also typically contain hydroxyproline, hydroxylysine,and desmosine. Type I, Type V, and Type X collagen are also typical ineggshell membrane.

Eggshell membrane also has been shown to contain acid glycosaminoglycansincluding dermatan sulfate and chondroitin-4-sulfate. Sulfatedglycoproteins have been isolated from eggshell membrane. Glycoproteinsin eggshell membrane include, for example, hexosamines, hexoses, andfucose.

In addition, hyaluronic acid has been detected in eggshell membrane.Other components identified in eggshell membrane include ovotransferrin,desmosine and isodesmosine, lysyl oxidase, and lysozyme.

In one embodiment, the naturally occurring material derived fromeggshell membrane is an eggshell membrane powder. For example, theeggshell membrane material may be dehydrated to produce eggshellmembrane flakes, which are then pulverized to produce an eggshellmembrane powder having a particle size between 100-500 microns.Powdering or pulverizing methods are known in the art, such as, forexample, by use of a standard milling or pulverizing procedure to treateggshell membrane flakes containing about 10% moisture.

In another embodiment, the naturally occurring material derived fromeggshell membrane is an eggshell membrane extract. In yet anotherembodiment, the naturally occurring material derived from eggshellmembrane is an eggshell membrane hydrolysate. In a further embodiment,the naturally occurring material derived from eggshell membrane is aneggshell membrane isolate.

Eggshell membrane, processed eggshell membrane, and eggshell membranehydrolysates or isolates contain naturally occurring materials derivedfrom eggshell membrane. These materials can be selected from the groupconsisting of a hyaluronic acid, hexosamine, chondroitin sulfate,collagen and combinations thereof.

The eggshell membrane, processed eggshell membrane, and eggshellmembrane hydrolysates and isolates are preferably free of any animalbody components or traces thereof, e.g., animal tissue, blood or bodyfluid components, which are detrimental or undesirable for thecontemplated use of the naturally occurring material or composition.

Although certain embodiments or examples may be described herein withreference to eggshell membrane, one skilled in the art can ascertain useof fowl eggshells. For example, one skilled in the art can ascertainvarious methods for processing of eggshells to obtain, extract, and/orpurify the naturally occurring materials (i.e., hyaluronic acid,glucosamine, chondroitin sulfate, collagens) from eggshell sources. Suchequivalents are intended to be encompassed in the scope of the presentinvention.

EXAMPLES

The following non-limiting examples have been carried out to illustrateembodiments of the invention.

Example 1 Preparation of Eggshell Membrane Flakes and Powder

The following example relates to the preparation of eggshell membraneflakes and powder. Hen eggshells and attached eggshell membrane wereobtained from an egg breaking facility. The eggshell membrane was firstseparated from eggshells. Eggshell membrane flakes were collected andimmediately packaged in plastic bags and placed in storage. Powderingwas accomplished using standard milling or pulverizing procedures totreat eggshell membrane flakes containing about 10% moisture. The powderwas subsequently sized by screening the pulverized powder through aseries of calibrated screens to produce a particle size range from100-500 microns.

Example 2 Treatment of a Disease Associated with Abnormal GlucoseMetabolism in an Insulin-Dependent Diabetic Human

Eggshell membrane powder capsules containing about 500 mg of eggshellmembrane powder were prepared as described in U.S. patent applicationSer. No.11/943,169. The capsule was administered orally to aninsulin-dependent diabetic human once before every meal, daily. Afterabout 90 days, the patient experienced greater blood sugar control withaverage fasting blood glucose decreasing from 120-130 mg/dL to 110-120mg/dL. Because the patient was insulin-dependent, hemoglobin A1c waswell-controlled in the 6.0-6.5% range. The decrease in fasting bloodglucose continued over time and the patient found it necessary todecrease their insulin intake accordingly to avoid fasting hypoglycemia.After about 18 months, the patient was no longer insulin-dependent andwas no longer required to take insulin injections nor any oral diabeticmedication, other than the eggshell membrane capsule. The patient is nowable to control their fasting blood glucose levels through diet andexercise and has normal hemoglobin A1c levels, i.e., less than about6.5% total hemoglobin.

Example 2 Reduction of Incidence or Progression of Insulin-DependentDiabetes Mellitus in a Pre-Diabetic Patient

Eggshell membrane powder prepared as in Example 1 was placed in capsulescontaining 500 mg of eggshell membrane powder. The capsule wasadministered orally to the insulin-dependent diabetic human once beforeevery meal, daily. After about one year, the patient observed a fastingblood glucose that is less than about 120 mg/dL.

Example 3 Method of Identifying Candidate Compound or Candidate Mixtureof Compounds in Eggshell, Eggshell Membrane, or a Combination ThereofWhich Treats and/or Reduces Incidence of or Progression of a Disease orCondition Associated with Abnormal Glucose Metabolism

The term “candidate compound” refers to any compound for which evidenceof treating and/or reducing incidence of or progression of a disease orcondition associated with abnormal glucose metabolism. The term“candidate mixture of compounds” refers to any combination of two and/ormore compounds for which evidence of treating and/or reducing incidenceof or progression of a disease or condition associated with abnormalglucose metabolism exists.

Examples of compounds include biological molecules and small molecules.Such evidence includes, for example, evidence that the compound ormixture of compounds treats and/or reduces incidence of or progressionof a disease or condition associated with abnormal glucose metabolism ina mammal.

A biological molecule is any molecule which contains more than onenucleotide, saccharide, or an amino acid unit, and has a molecularweight greater than about 450. Molecules that contain more than onenucleotide units include nucleic acids, oligonucleotides andpolynucleotides. Molecules that contain more than one saccharide unitinclude disaccharides, trisaccharides, oligosaccharides (more than foursaccharides) and polysaccharides. Molecules that contain more than oneamino acid units include oligopeptides, peptides, proteins, andpolypeptides.

Biological molecules further include derivatives of any of the moleculesdescribed above. For example, derivatives of biological moleculesinclude lipid and glycosylation derivatives of molecules that containmore than one amino acid unit, e.g., lipoproteins and glycoproteins.Derivatives of biological molecules further include lipid andglycosylated derivatives of molecules that contain more than onesaccharide unit, e.g. lipopolysaccharides and glycopolysaccharides.Derivatives of biological molecules further include proteo-derivativesof molecules that contain more than one nucleotide units.

Small molecules are typically organic compounds, includingorganometallic and organosilicon compounds, and the like, and generallyhave molecular weights of approximately 450 or less. Small molecules canfurther include molecules that would otherwise be considered biologicalmolecules, except their molecular weight is not greater thanapproximately 450. Thus, small molecules can include monosaccharides,oligosaccharides, amino acids, oligopeptides, nucleotides,oligonucleotides, and their derivatives, having a molecular weight ofapproximately 450 or less.

A small molecule can have any molecular weight. They are merely calledsmall molecules because they do not qualify as biological molecules, andtypically have molecular weights less than approximately 450.

The method for identifying a candidate compound or candidate mixture ofcompounds includes separating an eggshell membrane from an egg white andeggshell using any suitable mechanical and/or chemical technique knownin the art.

The eggshell membrane may be subjected to a solubilization process forsolubilizing at least one biological molecule or small molecule from theeggshell membrane. The eggshell membrane may also be subjected tohydrolysis by a suitable means known in the art.

The resulting solubilized or hydrolyzed components can be additionalmethods to further purify, isolate, and/or concentrate the components.For example, once the proteinaceous material or compounds aresolubilized or hydrolyzed, one skilled in the art can use standardbiochemistry methods to isolate a protein or compound of interest.Examples of such methods include all types of chromatography (e.g., highpressure liquid chromatography (HPLC) and column chromatography),fractional distillation, and extracting techniques using varioussolvents. Examples of useful solvents include water, alcohols (e.g.,methanol, ethanol, butanol, and propanol), dimethyl sulfoxide, dimethylformamide, tetrahydrofluan, hexane, ethyl acetate, and chloroform,and/or any mixtures thereof.

The method for identifying a candidate compound or candidate mixture ofcompounds further includes contacting the compound or mixture ofcompounds with a gastrointestinal tract cell. The gastrointestinal cellcan be any cell type found in the gastrointestinal tract, including, forexample, epithelial cells, endothelial cells, smooth muscle cells,endocrine cells, secretory cells, mucosal cells, and absorptive cells ofthe intestinal epithelium. Gastrointestinal tract cells include, forexample, a tonsil cell, esophageal cell, stomach cell, pancreatic cell,pancreatic beta cell, colonic cell, intestinal cell (e.g., smallintestinal cell and large intestinal cell), as well as a distal ileumcell. The contacting may be performed via in vitro or in vivo methods.

In vitro methods typically include mixing the compound or mixture ofcompounds with suitable cells in a culture medium. The order of addingthe compound or mixture of compounds and the cells to the culture mediumis not critical. The gastrointestinal tract cells may be independent ofother cells, or may be associated with other cells.

In vivo methods typically involve the administration of the compound ormixture of compounds, such as those described above, to thegastrointestinal cell of a mammal, preferably a human or laboratorymammal. The compounds or mixture of compounds useful in the methods ofthe present invention are administered to a mammal in an amount that,for example, decreases hemoglobin A1c levels or decreases fasting bloodglucose concentration. The administered amount may vary over a largerange, and may be determined during pre-assay tests by methods familiarto one of ordinary skill in the art.

The compound or mixture of compounds useful in the methods of thepresent invention may be administered to a mammal by any of a number ofwell-known methods for administering compounds or mixture of compounds.For example, the compound or mixture of compounds may be administeredorally (including to laboratory animals by gavage), sublingually,parenterally, enterally, rectally, by suppository, buccally,intravenously, intranasally, intramuscularly, subcutaneously, and/ortransdermally. Other routes of administration include intubation, whichrefers to placement of a nasogastric feeding tube or a gastric feedingtube into the body of a laboratory animal.

After the compound or mixture of compounds has had time, for example, todecrease hemoglobin A1c levels or decrease fasting blood glucoseconcentration, the method further includes obtaining a biological samplefrom the mammal, and determining the candidate compound or a candidatemixture of compounds that treats and/or reduces incidence of orprogression of a disease or condition associated with abnormal glucosemetabolism in a mammal.

1. A method for treating a disease or condition associated with abnormal glucose metabolism, said method comprising orally administering to a mammal in need thereof an effective amount of a composition comprising powdered eggshell membrane.
 2. The method of claim 1, wherein the disease or condition is insulin resistance.
 3. The method of claim 1, wherein the disease or condition is glucose intolerance
 4. The method of claim 1, wherein the disease or condition is glucose non-responsiveness.
 5. (canceled)
 6. The method of claim 1, wherein the effective amount is effective to achieve at least a 0.2 percentage point decrease in hemoglobin A1c.
 7. The method of claim 1, wherein the effective amount is effective to achieve at least a 1 percentage point decrease in hemoglobin A1c.
 8. The method of claim 1, wherein the effective amount is effective to achieve at least a 2 percentage point decrease in hemoglobin A1c.
 9. The method of claim 1, wherein the effective amount is effective to achieve at least a 3 percentage point decrease in hemoglobin A1c.
 10. The method of claim 1, wherein the effective amount is effective to decrease a fasting blood glucose concentration in said mammal by at least 5 mg/dL.
 11. The method of claim 1, wherein the effective amount is effective to decrease a fasting blood glucose concentration in said mammal by at least 10 mg/dL.
 12. The method of claim 1, wherein the effective amount is effective to decrease a fasting blood glucose concentration in said mammal by at least 20 mg/dL.
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. The method of claim 1, wherein the naturally occurring material is water soluble.
 17. The method of claim 1, wherein the naturally occurring material is water insoluble.
 18. The method of claim 1, wherein the composition comprising powdered eggshell membrane comprises the following in weight percent: a. about 0.25 to about 5 wt % hexosamine; b. about 0.3 to about 3 wt % glycosaminoglycan; c. about 0.5 to about 5 wt % hyaluronic acid; d. about 0.01 to about 2 wt % sialic acid; and/or e. about 5 to about 20 wt % collagen.
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. The method of claim 1, wherein the mammal is insulin-dependent.
 24. The method of claim 1, wherein the mammal has a fasting blood glucose concentration at least 100 mg/dL and at most 125 mg/dL.
 25. (canceled) 